The histones, histone variants, and high mobility group (HMG) proteins have roles in chromatin structure and function that are applicable to all eukaryotic cells and some roles that are specific to the process of cell differentiation during spermatogenesis. In order to elucidate their function, a better understanding of the characteristics and transitions of these chromosomal proteins in spermatogenic cells is needed. Towards this end, we will first further characterize the histone variants, post-synthetic modifications of histones, and HMG variants that are present in rat spermatogenic cells. Next, we will localize these proteins, their synthesis, and their modifications to specific stages of spermatogenesis by biochemical analysis of purified populations of rat testis cells or nuclei. We will employ methods we have already developed, including elutriation and density separation in Percoll gradients, to isolate pachytene spermatocytes and spermatids, and we will also develop new methods for separation of early primary spermatocytes, spermatogonia, and gonocytes. The effects of these variant and modified proteins on chromatin structure and function will be studied by examining the distribution of proteins in fractions of nuclease digested chromatin, in specific nucleosomal subclasses, and by chemical crosslinking of histones. The above experiments should provide tests for the validity of suggested roles for histone (and HMG) variants and modification in the dramatic chromosomal events that occur during spermatogenesis. These possibilities will be further tested by comparison of histone metabolism in normal rat spermatocytes, in rat oocytes, and in spermatocytes perturbed with 5-flurouracil.